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Table S1. Primer sequences for the determination of expression of metabolic genes. Primers for quantitative PCR analysis of expression levels of metabolic genes. (DOCX 14 kb)

Figure S1. No microgliosis in both Cre-positive and Cre-negative control mice. (A-D) No differences in microglia number and shape are observed between Cre-positive (Cre Mfp2Wt/LoxP) and Cre-negative (Mfp2Wt/LoxP) control mice at 5 months of age (A-D) and 12 months of age (not shown). Representative pictures are shown. n = 3–5 mice/group. (PPTX 988 kb)

Figure S3. Inflammatory properties of cultured Mfp2−/− and control microglia. MACS-isolated microglia from P8 mice were kept either in basal conditions or polarized to a pro-inflammatory state (Il1β/IFNγ) or an anti-inflammatory state (IL4). Transcript expression of pro-inflammatory (Tnfa, iNOS, Cxcl1) and anti-inflammatory cytokines (Arg1, Fizz1, Ym1) were determined. Significance levels: Φ p

Additional file 2: Figure S1. Poly(GA) pathology in the pineal gland of C9orf72 and nonC9orf72 ALS and FTLD cases. Figure S2. VIP-immunostaining of the SCN-related neurons in C9orf72 and nonC9orf72 ALS and FTLD cases.

Figure S2. Efficient and selective inactivation of MFP2 in microglia in Cx3cr1-Mfp2−/− mice. (A) Microglia were isolated from 11-month-old control and Cx3cr1-Mfp2−/− mice, and microglia purity was confirmed by the high expression of microglial markers (Tmem119 and P2ry12) in the positive (microglia) versus the negative fraction (neurons, astrocytes, and oligodendrocytes). Transcript expression of Mfp2 was determined in control and Cx3cr1-Mfp2−/− in both positive and negative fraction. Representative experiment out of two with similar results. (B) MFP2 activity in brain homogenates of control and Cx3cr1-Mfp2−/− mice. n = 2–3 mice/group. Mean ± SD is shown. (PPTX 46 kb)

Figure S1. In vitro and in vivo pharmacokinetic properties of ACY-738. (A) Western blots showing the effects of different concentrations of ACY-738 on acetylation of α-tubulin and histone 3 in N2a cells. (B) Dose-response curves of the effect of ACY-738 based on the quantification of Western blots. The ratio of acetylated α-tubulin is normalized to calnexin (top) and acetylation of histone 3 is normalized to histone 4 (bottom). Values were normalized to vehicle (n = 2). (C) Dose-response curve showing the effect of ACY-738 on acetylation of histone 3 in nuclear fractions of spinal cord lysates of non-Tg mice using a colorimetric HDAC activity kit. The orange band depicts the concentration range of the compound as measured in tissues of treated Tg FUS+/+ mice. (D) Concentrations of ACY-738 in the plasma and brain of Tg FUS+/+ mice measured by mass spectrometry. Half maximal inhibitory concentration (IC50) (n = 4). (PDF 73 kb)

Figure S5. Transcriptomics and proteomics analyses show a high degree of similarity. (A) Multidimensional scaling (MDS) of transcriptomics data. (B) Heat map by hierarchical clustering of all differentially expressed genes. (C) Venn diagram representing the overlap of differentially expressed genes between vehicle-treated Tg FUS+/+ mice and non-Tg controls (blue), between ACY-738-treated Tg FUS+/+ mice and non-Tg controls (light orange) and between vehicle- and ACY-738-treated Tg FUS+/+ mice (dark orange). (D) MDS of proteomics data. (E) Heat map by hierarchical clustering of all differentially expressed proteins. (F) Venn diagram representing the overlap of differentially expressed proteins between vehicle-treated Tg FUS+/+ mice and non-Tg controls (blue), between ACY-738-treated Tg FUS+/+ mice and non-Tg controls (light orange) and between vehicle- and ACY-738-treated Tg FUS+/+ mice (dark orange). (G) Venn diagram comparing differential expression in vehicle-treated Tg FUS+/+ mice compared to non-Tg controls on the mRNA- (blue grid) and protein-level (blue lines) of all identified mRNA-protein pairs. (H) Correlation plot of all identified mRNA-protein pairs, comparing the expression values on the mRNA and protein level in vehicle-treated Tg FUS+/+ mice compared to non-Tg controls. (I) Correlation plot of genes that were differentially expressed in both data sets, comparing the expression values on the mRNA and protein level in vehicle-treated Tg FUS+/+ mice compared to non-Tg controls. (J) Venn diagram comparing differential expression in ACY-738-treated Tg FUS+/+ mice compared to non-Tg controls on the mRNA- (orange grid) and protein-level (orange lines) of all identified mRNA-protein pairs. (K) Correlation plot of all identified mRNA-protein pairs, comparing the expression values on the mRNA and protein level in ACY-738-treated Tg FUS+/+ mice compared to non-Tg controls. (L) Correlation plot of genes that were differentially expressed in both datasets, comparing the expression values on the mRNA and protein level in ACY-738-treated Tg FUS+/+ mice compared to non-Tg controls. (PDF 225 kb)

Figure S4. ACY-738 does not affect gliosis in the spinal cord of Tg FUS+/+ mice. Immunostaining for astrogliosis and microgliosis in the ventral horn of the lumbar spinal cord of P60 non-Tg controls, vehicle- and ACY-738-treated Tg FUS+/+ mice. GFAP was used as a marker for astrogliosis, CD11b as a marker for microgliosis. Scale bar = 100 μm. (PDF 4055 kb)

Genetic variants identified in patients with SLONM using NGS and a panel of 283 genes. (DOC 72 kb)

Genetic variants identified in patients with SLONM using NGS and a panel of 283 genes. (DOC 72 kb)

Figure S6. The expression of metabolic genes is partially restored by ACY-738 therapy. Heatmap of expression changes of a subset of metabolic genes at the mRNA and protein level based on transcriptomics and proteomics experiments. Hexokinase 2 (Hk2), pyruvate dehydrogenase kinase 4 (Pdk4), Acyl-CoA synthetase long-chain family member 6 (Acsl6), acyl-CoA dehydrogenase family member 11 (Acad11), 3-oxoacid CoA-transferase (Oxct1), 7-Dehydrocholesterol reductase (7-Dhcr), squalene epoxidase (Sqle), Elongation of very long chain fatty acids protein 7 (Elovl7), 1-acylglycerol-3-phosphate O-acyltransferase 4 (Agpat4), Apolipoprotein D (Apod), Apolipoprotein E (Apoe), Fatty acid binding protein 4 (Fabp4). (PDF 62 kb)

Figure S6. The expression of metabolic genes is partially restored by ACY-738 therapy. Heatmap of expression changes of a subset of metabolic genes at the mRNA and protein level based on transcriptomics and proteomics experiments. Hexokinase 2 (Hk2), pyruvate dehydrogenase kinase 4 (Pdk4), Acyl-CoA synthetase long-chain family member 6 (Acsl6), acyl-CoA dehydrogenase family member 11 (Acad11), 3-oxoacid CoA-transferase (Oxct1), 7-Dehydrocholesterol reductase (7-Dhcr), squalene epoxidase (Sqle), Elongation of very long chain fatty acids protein 7 (Elovl7), 1-acylglycerol-3-phosphate O-acyltransferase 4 (Agpat4), Apolipoprotein D (Apod), Apolipoprotein E (Apoe), Fatty acid binding protein 4 (Fabp4). (PDF 62 kb)